SUMMARY After primary infection, neurotropic polyomavirus JC (JCV) enters a persistent/latent state through the action of the immune system. Immune dysfuction plays a critical role in the reactivation of JCV in brain and development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). Once a rare disease primarily affecting lymphoma/leukemia patients and transplant recipients, PML is now seen more often due to the AIDS pandemic and the introduction of immunomodulatory drugs for the treatment of autoimmune disorders including multiple sclerosis (MS) and Crohn's disease (CD). Under these conditions, expression of the JCV genome and its replication in glial cells of the CNS including astrocytes and oligodendrocytes, the myelin producing cells in the brain, leads to white matter demyelination. Evidently, cell type-specific transcription of the JCV early gene by glial specific transcription factors and production of the viral early protein, T-antigen, the critical protein that initiates a cycle of productive viral infection leading to cytolytic destruction of oligodendrocytes and the formation of bizarre dysfunctional astrocytes. Currently, there is no effective strategy for inhibition of JCV replication and PML remains an incurable and often fatal disease. Recently, we have employed the CRISPR/Cas9 system in single and multiplex configurations specific to JCV that compromise the integrity of the viral DNA sequences corresponding to the various regions of DNA encoding T-antigen by introducing insertion/deletion (InDel) mutations. Our preliminary results show that JCV- specific gene editing results in the inactivation of viral gene expression and replication in glial cells. Our methods for disrupting the JCV genome exhibit no cytotoxicity/genotoxicity to host cells. Thus, our CRISPR- Cas9 system has the potential to remove a large segment of the JCV genome and cripple the ability of the virus to replicate in infected cells. Furthermore, our preliminary data also suggest that the pre-existing presence of JCV-directed Cas9 in cells protected them against JCV infection. Based on these preliminary observations, we propose to develop an RNA-guided Cas9 gene editing platform that acts as molecular scissors and, by disrupting various regions of the JCV genome, including the regulatory domain, abrogates replication of the virus in glial cells. The outcome of these proof-of-concept molecular, genetic, and virological studies will set the stage for a more comprehensive initiative for developing a novel strategy and examining` the efficacy of this system in pre-clinical and clinical settings for the treatment of PML.